We aim to continue our studies of the pioneer translation initiation complex and the pioneer round of translation. Nonsense-mediated mRNA decay (NMD) is an important quality control mechanism that eliminates transcripts having the potential to generate truncated proteins that could be deleterious to cells. We have found that NMD in mammalian cells generally occurs when translation terminates more than -50- 55 nts upstream of a splicing-generated exon-exon junction during a pioneer round of translation. By definition, this round of translation utilizes mRNA that is bound by the cap binding protein heterodimer CBP80/20. The role of the exon-exon junction in NMD reflects the post-splicing deposition of an exon junction complex (EJC) of proteins that include the Upf NMD factors. We have found that EJCs typify spliced CBP80/20-bound mRNA but not its remodeled product, elF4E-bound mRNA. To date, we have shown that the pioneer translation initiation complex is functionally distinct from but structurally overlaps with the steady- state, i.e., elF4E-bound, translation initiation complex. We have identified degradative activities involved in NMD. We also began to unravel why some mRNAs are subject to nucleus-associated NMD whereas other mRNAs are subject to cytoplasmic NMD. In Aim 1, we will study further the structure of the pioneer translation initiation complex before and during translation, and we will characterize the interacting domains of CBP80-Upf 1, CBP80-SMG1, CBP80-elF4GI, Upf 1 -eRF1 and Upf 1 -eRF3 that we have shown exist. In Aim 2, we will determine why some mRNAs are targeted for nucleus-associated NMD and other mRNAs are targeted for cytoplasmic NMD and, in collaboration with Rob Singer's lab, we will localize the cellular site of nucleus-associated NMD using fluorescent in situ hybridization of single RNA molecules. In Aim 3, we will solidify data obtained in collaboration with Ben Blencowe's lab indicating that there are functional differences among different EJCs or if any EJC that resides sufficiently downstream of a nonsense codon can trigger NMD. These differences will be exploited to determine if NMD is triggered by only the 3'-most EJC or if any EJC that resides more than 50-55 nucleotides downstream of a nonsense codon can trigger NMD. We expect that renewed support of this grant will allow us to continue to make major advances in understanding the mechanism of NMD in mammalian cells.